WO2016036209A1 - Microorganisme ayant une productivité améliorée de l-thréonine, et procédé de production de l-thréonine au moyen de celui-ci - Google Patents

Microorganisme ayant une productivité améliorée de l-thréonine, et procédé de production de l-thréonine au moyen de celui-ci Download PDF

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WO2016036209A1
WO2016036209A1 PCT/KR2015/009381 KR2015009381W WO2016036209A1 WO 2016036209 A1 WO2016036209 A1 WO 2016036209A1 KR 2015009381 W KR2015009381 W KR 2015009381W WO 2016036209 A1 WO2016036209 A1 WO 2016036209A1
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amino acid
substituted
rpod
threonine
amino
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PCT/KR2015/009381
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English (en)
Korean (ko)
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이지선
이광호
김효진
이근철
황영빈
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씨제이제일제당 주식회사
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Priority to CN201580006567.2A priority Critical patent/CN106029879B/zh
Priority to BR112016015218-2A priority patent/BR112016015218B1/pt
Priority to PL15837846T priority patent/PL3144385T3/pl
Priority to ES15837846T priority patent/ES2820583T3/es
Priority to US15/117,437 priority patent/US10760108B2/en
Priority to EP15837846.3A priority patent/EP3144385B1/fr
Priority claimed from KR1020150125440A external-priority patent/KR101865998B1/ko
Publication of WO2016036209A1 publication Critical patent/WO2016036209A1/fr
Priority to US16/447,814 priority patent/US10968467B2/en
Priority to US16/930,082 priority patent/US11312982B2/en

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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C12N9/10—Transferases (2.)
    • C12N9/12—Transferases (2.) transferring phosphorus containing groups, e.g. kinases (2.7)
    • C12N9/1241—Nucleotidyltransferases (2.7.7)
    • C12N9/1247—DNA-directed RNA polymerase (2.7.7.6)
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/195—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from bacteria
    • C07K14/24—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from bacteria from Enterobacteriaceae (F), e.g. Citrobacter, Serratia, Proteus, Providencia, Morganella, Yersinia
    • C07K14/245—Escherichia (G)
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C12N9/10—Transferases (2.)
    • C12N9/12—Transferases (2.) transferring phosphorus containing groups, e.g. kinases (2.7)
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
    • C12P13/00—Preparation of nitrogen-containing organic compounds
    • C12P13/04—Alpha- or beta- amino acids
    • C12P13/08—Lysine; Diaminopimelic acid; Threonine; Valine
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Y—ENZYMES
    • C12Y207/00—Transferases transferring phosphorus-containing groups (2.7)
    • C12Y207/07—Nucleotidyltransferases (2.7.7)
    • C12Y207/07006—DNA-directed RNA polymerase (2.7.7.6)

Definitions

  • the present invention relates to a novel variant RNA polymerase sigma factor 70 ( ⁇ 70 ) polypeptide, a polynucleotide encoding the same, a microorganism comprising the polypeptide and a method for producing L-threonine using the microorganism.
  • Useful products such as amino acids can be produced by fermentation using strains developed by artificial variation or genetic recombination. In particular, if the genetic factors found directly or indirectly involved in the upper stage for the development of these strains for mass production, and can be used properly, strains with higher yields can be developed.
  • a representative technique is gTME (global transcription machinery engineering) technology that regulates the expression of all genes in a cell by randomly mutating the recruiting protein of RNA polymerase.
  • RNA polymerase is a macromolecule consisting of five subunits and consists of two alpha ( ⁇ ), beta ( ⁇ ), beta prime ( ⁇ ') and omega ( ⁇ )
  • the enzyme (holoenzyme) is represented by ⁇ 2 ⁇ ' ⁇ .
  • Sigma factor ( ⁇ factor) together with these complete enzymes, is a prokaryotic transcription initiation factor that enables specific binding of RNA polymerase to promoters of genes and is distinguished by molecular weight.
  • ⁇ 70 refers to a sigma factor with a molecular weight of 70 kDa (Gruber TM, Gross CA, Annu Rev Microbiol. 57: 441-66, 2003).
  • Escherichia coli includes housekeeping sigma factor ⁇ 70 (RpoD), nitrogen-restricted sigma factor ⁇ 54 (RpoN), starvation / stationary phase sigma factor ⁇ 38 (RpoS), and heat shock. ) Sigma factor ⁇ 32 (RpoH), flagella sigma factor ⁇ 28 (RpoF), extracellular / extreme heat stress sigma factor ⁇ 24 (RpoE), ferric citrate sigma factor ⁇ 19 (FecI) and the like are known. These different sigma factors are known to be activated under different environmental conditions, and these specialized sigma factors combine with the promoters of native genes that are transcribed under specific circumstances to regulate their transcription.
  • the present inventors have made intensive efforts to develop microorganisms that produce higher concentrations of L-threonine without inhibiting the growth of host cells.
  • a sigma factor 70 polypeptide of a novel variant RNA polymerase to produce L-threonine.
  • the present invention was completed by confirming that a strain having improved ability to produce L-threonine can be developed by introducing into Escherichia.
  • Another object of the present invention is to provide a polynucleotide encoding the polypeptide.
  • Another object of the present invention is to provide a microorganism transformed to include the polypeptide.
  • Another object of the present invention is to obtain a culture by culturing the microorganism; And it provides a method for producing L-threonine comprising the step of recovering L-threonine from the culture or microorganism.
  • the present invention can identify novel variants of the polypeptide of RNA polymerase sigma factor 70 that can upregulate L-threonine production capacity.
  • the microorganism expressing the mutant polypeptide on the basis of the production yield of L-threonine is remarkably excellent, it can be expected to reduce the production cost and the convenience of production in the industrial aspect.
  • the present invention provides a variant polypeptide having novel RNA polymerase sigma factor 70 activity.
  • RNA polymerase sigma factor 70 is a transcription initiation factor that works with RNA polymerase, which corresponds to one of the sigma factors ( ⁇ 70 ) and is also called sigma factor D (RpoD). do. Sigma factors are involved in transcriptional regulation by interacting with upstream DNA (UP element) and various transcriptional regulators upstream of a specific promoter.
  • sigma factor 70 ( ⁇ 70 ) is the main regulator of most of the house keeping genes and key genes of E. coli sigma, and is a sigma factor that dominates during the growth of the exponential phase. (Jishage M, et al, J Bacteriol 178 (18); 5447-51,1996).
  • ⁇ 70 protein may include the amino acid sequence of SEQ ID NO: 8, but is not limited thereto as long as it has the same activity as ⁇ 70 of the present invention.
  • variant polypeptide refers to a part or whole of the amino acid sequence of a wild-type polypeptide.
  • a part of the amino acid sequence of a polypeptide having RNA polymerase sigma factor 70 ( ⁇ 70 ) activity is replaced by a wild type ( It is a polypeptide having an RNA polymerase sigma factor 70 ( ⁇ 70 ) activity having a sequence different from the wild-type amino acid sequence. That is, the present invention provides a sigma factor 70 ( ⁇ 70 ) mutant polypeptide that contributes to the improvement of L-threonine production, rather than the wild type sigma factor 70 ( ⁇ 70 ) polypeptide.
  • the variant polypeptide is a variant polypeptide having RNA polymerase sigma factor 70 activity in which at least one amino acid of the amino acid sequence of SEQ ID NO: 8 is substituted with another amino acid in the polypeptide consisting of SEQ ID NO: 8, wherein the variant position Is the first methionine as the first amino acid from 440 to 450 amino acids therefrom; 459th amino acid; 466th amino acid; Amino acids 470-479; 484 th amino acid; 495-499 amino acids; 509th amino acid; 527th amino acid; 565th to 570th amino acids; 575th to 580th amino acids; 599th amino acid; And 612th amino acid.
  • the variant polypeptide has 45 mutation positions (440 to 450, 459, 466, 470 to 479, 484, 495 to 499, 509, 527, 565 to 570, and 575).
  • 1 to 580, 599, 612) may be a polypeptide substituted at one or more positions with another amino acid.
  • one, two, three, four, five, six, seven, eight, nine, ten or more but RNA polymerase contributes to improved L-threonine production capacity It may be included without limitation as long as it has sigma factor 70 activity.
  • the 440 th to 450 th amino acids 440 th amino acid, 446 th amino acid or 448 th amino acid;
  • the 470 th to 479 th amino acids a 474 th amino acid or a 477 th amino acid; 496 th or 498 th amino acids of the 495 th to 499 th amino acids; 567 th or 569 th amino acids, among the 565 th to 570 th amino acids;
  • the 576 th amino acid or the 579 th amino acid may be substituted with another amino acid, but is not limited thereto.
  • More specific amino acid substitutions include replacement of the 440th amino acid with proline from the starting methionine (T440P); 446th amino acid is substituted with proline (Q446P); 448th amino acid is substituted with serine (R448S); 459th amino acid is substituted with asparagine (T459N); 466th amino acid is substituted with serine (I466S); 474th amino acid is substituted with valine (M474V); 477th amino acid is substituted with glycine (E477G); 484th amino acid is substituted with valine (A484V); 496th amino acid is substituted with asparagine (K496N); 498th amino acid is substituted with arginine (L498R); 509th amino acid is substituted with methionine (T509M); 527th amino acid substituted by proline (T527P); 567th amino acid is substituted with valine (M567V); 569th amino acid is substituted with proline
  • the variant polypeptide may be a polypeptide having an amino acid sequence of any one of the amino acid sequences of SEQ ID NOs: 9 to 37, but is not limited thereto.
  • Variant polypeptides of the present invention in addition to the amino acid sequence set forth in SEQ ID NOs: 9-37, 70% or more, specifically 80% or more, more specifically 90% or more, more specifically 99% or more of the sequence
  • amino acid sequence showing homology any protein that contributes to the improvement of L-threonine production ability as compared to the wild type sigma factor 70 ( ⁇ 70 ) protein can be included without limitation.
  • sequence having such homology is an amino acid sequence having the same or corresponding biological activity as that of the mutant sigma factor 70 ( ⁇ 70 ) protein, even if some sequences have an amino acid sequence deleted, modified, substituted or added, It is obvious that it is included in the scope of the invention.
  • the term “homology” refers to the same degree of base or amino acid residues between sequences in an amino acid or nucleotide sequence of a gene encoding a protein. do. If the homology is sufficiently high, the expression products of the gene of interest may have the same or similar activity.
  • the percent sequence identity can be determined using known sequence comparison programs, and examples include BLAST (NCBI), CLC Main Workbench (CLC bio), MegAlign TM (DNASTAR Inc), and the like.
  • Another aspect of the invention provides a polynucleotide encoding said variant polypeptide.
  • polynucleotide is a polymer of nucleotides in which nucleotide monomers are long chained by covalent bonds, and are DNA or RNA strands of a predetermined length or more, and more specifically, the variant polypeptide Means the polynucleotide fragment to be encoded.
  • the gene encoding the amino acid sequence of the RNA polymerase sigma70 factor is rpoD gene, specifically from the genus Escherichia, more specifically Escherichia coli have.
  • the polynucleotide encoding the wild-type RNA polymerase sigma factor 70 may be represented by SEQ ID NO: 7, but is not limited thereto.
  • the genetic code degeneracy genetic code degeneracy
  • the base sequence encoding the same amino acid sequence and variants thereof are also included in the present invention.
  • nucleotide sequence encoding the same amino acid sequence and variants thereof are also included in the present invention due to the degeneracy of the genetic code.
  • the polypeptide consisting of the amino acid sequence of SEQ ID NO: 8 may include a base sequence encoding the amino acid sequence of the polypeptide substituted with one or more amino acids of the amino acid at the following position and a variant thereof.
  • the mutation position is a starting methionine as the first amino acid from 440 to 450 amino acids therefrom; 459th amino acid; 466th amino acid; Amino acids 470-479; 484 th amino acid; 495-499 amino acids; 509th amino acid; 527th amino acid; 565th to 570th amino acids; 575th to 580th amino acids; 599th amino acid; And 612th amino acid.
  • the mutation position is the 440th amino acid is substituted with proline (T440P); 446th amino acid is substituted with proline (Q446P); 448th amino acid is substituted with serine (R448S); 459th amino acid is substituted with asparagine (T459N); 466th amino acid is substituted with serine (I466S); 474th amino acid is substituted with valine (M474V); 477th amino acid is substituted with glycine (E477G); 484th amino acid is substituted with valine (A484V); 496th amino acid is substituted with asparagine (K496N); 498th amino acid is substituted with arginine (L498R); 509th amino acid is substituted with methionine (T509M); 527th amino acid substituted by proline (T527P); 567th amino acid is substituted with valine (M567V); 569th amino acid is substituted with proline (T569P); 5
  • the base sequence encoding any one amino acid sequence of the amino acid sequence of SEQ ID NO: 9 to 37 and its variants may be included, but is not limited thereto.
  • the invention is a host cell comprising a polynucleotide encoding the variant polypeptide, a microorganism transformed with a vector comprising the polynucleotide encoding the variant polypeptide or the variant polypeptide
  • a host cell comprising a polynucleotide encoding the variant polypeptide, a microorganism transformed with a vector comprising the polynucleotide encoding the variant polypeptide or the variant polypeptide.
  • the introduction may be made by transformation, but is not limited thereto.
  • sigma factor 70 ( ⁇ 70) mutant microorganism containing a polypeptide-type production is enhanced ability of the wild-type sigma factor 70 ( ⁇ 70) as compared to microorganisms containing the polypeptides L- write the growth of the host cell without prejudice Leo Nin Therefore, L-threonine can be obtained in high yield from these microorganisms.
  • the term "vector” refers to any medium for cloning and / or transferring bases to a host cell.
  • the vector may be a replica that other DNA fragments can bind to and result in replication of the bound fragments.
  • "Replication unit” refers to any genetic unit (e.g., plasmid, phage, cosmid, chromosome, virus) that functions as a self-unit of DNA replication in vivo, i.e., is replicable by its own regulation. .
  • the "vector” includes viral and non-viral mediators for introducing bases into host cells in vitro, ex vivo or in vivo, and may also include minispherical DNA.
  • the vector may be a plasmid without a bacterial DNA sequence (Ehrhardt, A. et al. (2003) Hum Gene Ther 10: 215-25; Yet, NS (2002) MoI Ther 5: 731-38; Chen, ZY et al. (2004) Gene Ther 11: 856-64).
  • the vector may include a transposon (Annu Rev Genet. 2003; 37: 3-29.), Or an artificial chromosome.
  • pACYC177, pACYC184, pCL1920, pECCG117, pUC19, pBR322, pDZ, pCC1BAC and pMW118 vectors may be used, but is not limited thereto.
  • the term "transformation" is to introduce a gene into the host cell to be expressed in the host cell, the transformed gene can be expressed in the host cell, in addition to the chromosome insertion or chromosome of the host cell Anything located is included without limitation.
  • the gene may be introduced into a host cell in the form of an expression cassette, which is a polynucleotide construct containing all elements necessary for self-expression.
  • the expression cassette may include a promoter, a transcription termination signal, a ribosomal binding site, and a translation termination signal that are usually operably linked to the gene.
  • the expression cassette may be in the form of an expression vector capable of self replication.
  • the gene may be introduced into the host cell in the form of a polynucleotide structure itself or operably linked to a sequence required for expression in the host cell, but is not limited thereto.
  • the term "host cell” or "microorganism” may include a polynucleotide encoding a variant polypeptide, or may be transformed with a vector comprising a polynucleotide encoding a variant polypeptide to express the variant polypeptide.
  • the host cell or microorganism may be any microorganism capable of producing L-threonine, including a sigma factor 70 ( ⁇ 70 ) mutant polypeptide.
  • Escherichia Escherichia
  • Serratia marcescens Serratia
  • An air Winiah Erwinia
  • Enterobacter bacteria Enterobacter bacteria
  • Salmonella Salmonella
  • Streptomyces Streptomyces
  • Pseudomonas Pseudomonas
  • Microorganism strains such as genus, Brevibacterium genus or Corynebacterium genus may be included, specifically Escherichia genus microorganisms, more specifically may be Escherichia coli
  • the present invention is not limited thereto.
  • the present invention provides a method for producing L-threonine, comprising culturing the described microorganisms in a medium and recovering L-threonine from the cultured microorganism or culture medium. Provide a method.
  • the term "culture” means to grow the microorganisms under appropriately artificially controlled environmental conditions. Cultivation process of the present invention can be made according to the appropriate medium and culture conditions known in the art. Conditions such as specific incubation temperature, incubation time and pH of the medium may be carried out according to the general knowledge of a person skilled in the art or a method known in the art, and can be adjusted accordingly. Specifically, these known culture methods are described in Chmiel; Bioreatechnik 1. Einbigung indie Biovonstechnik (Gustav Fischer Verlag, Stuttgart, 1991), and Storhas; Bioreaktoren und periphere bamboo (Vieweg Verlag, Braunschweig / Wiesbaden, 1994). In addition, the culture method may include a batch culture, a continuous culture (cintinuous culture) and fed-batch culture, specifically, a batch process or injection batch or repeated batch batch (fed batch) or repeated fed batch process), but is not limited thereto.
  • the medium used for culturing should meet the requirements of the particular strain in an appropriate manner, and the carbon sources that can be used in the medium include glucose and carbohydrates such as glucose, saccharose, lactose, fructose, maltose, starch, cellulose, Oils such as soybean oil, sunflower oil, castor oil, coconut oil and the like, fatty acids such as palmitic acid, stearic acid, linoleic acid, alcohols such as glycerol, ethanol, organic acids such as acetic acid, and the like. These materials can be used individually or as a mixture, but are not limited to these.
  • Nitrogen sources that may be used may include peptone, yeast extract, gravy, malt extract, corn steep liquor, soybean wheat and urea or inorganic compounds such as ammonium sulfate, ammonium chloride, ammonium phosphate, ammonium carbonate and ammonium nitrate, and the like.
  • the nitrogen source may also be used individually or as a mixture, but is not limited thereto.
  • Personnel that may be used may include potassium dihydrogen phosphate or dipotassium hydrogen phosphate or the corresponding sodium-containing salts and the like.
  • the culture medium may contain a metal salt such as magnesium sulfate or iron sulfate required for growth.
  • essential growth substances such as amino acids and vitamins can be used.
  • suitable precursors to the culture medium may be used.
  • the raw materials described above may be added batchwise or continuously in a suitable manner to the culture during the culturing process, but are not limited thereto.
  • compounds such as ammonium hydroxide, potassium hydroxide, ammonia, phosphoric acid and sulfuric acid can be added to the culture in a suitable manner to adjust the pH of the culture.
  • antifoaming agents such as fatty acid polyglycol esters can be used to suppress bubble generation.
  • oxygen or oxygen-containing gas may be injected into the culture, or nitrogen, hydrogen or carbon dioxide gas may be injected without injecting gas to maintain anaerobic and unaerobic conditions.
  • the temperature of the culture is usually 27 ° C to 37 ° C, specifically 30 ° C to 35 ° C. The incubation period can continue until the desired amount of useful material is obtained, specifically 10 to 100 hours.
  • L-threonine may be released into the culture medium or may be contained in microorganisms.
  • PCR polymerase chain reaction
  • PCR for amplification of the rpoD gene was performed using primers of SEQ ID NOs: 1 and 2, with denaturation at 95 ° C. for 30 seconds, annealing at 56 ° C. for 30 seconds and elongation at 72 ° C. for 2 minutes.
  • the cycle consisted of 27 repetitions.
  • rpoD fragment DNA fragment of about 2.0 Kb (hereinafter referred to as “rpoD fragment”) by electrophoresis on a 0.8% agarose gel and eluted.
  • Primer number Base sequence SEQ ID NO: One 5'-TACTCAAGCTTCGGCTTAAGTGCCGAAGAGC-3 ' One 2 5'-AGGGCGAATTCCTGATCCGGCCTACCGATTA-3 ' 2
  • Copycontrol pCC1BAC vector (EPICENTRE (USA)) was obtained by electrophoresis on 0.8% agarose gel by treatment with HindIII , EcoRI , eluting, ligation with the obtained rpoD fragment and pCC1BAC-rpoD plasmid was produced.
  • PCR was performed using the gDNA obtained in Example 1 as a template to obtain approximately 1.5 kb of the DNA fragment including the promoter region of the E. coli W3110 rpoD to the BamHI site within the gene. PCR using the primers of SEQ ID NOS: 1 and 3, repeated 27 cycles consisting of 30 seconds denaturation at 95 °C, 30 seconds annealing at 56 °C and 1 minute 30 seconds elongation at 72 °C as in Example 1 .
  • the PCR product was digested with BamHI and HindIII , and 1.5Kb DNA fragments (hereinafter, referred to as "patial rpoD fragments") were electrophoresed on 0.8% agarose gel and eluted.
  • Copycontrol pCC1BAC vector was treated with HindIII , BamHI , eluted by electrophoresis on a 0.8% agarose gel, and the resulting patial rpoD fragment was ligated to prepare a pCC1BAC-patial rpoD plasmid.
  • Example 1 the gDNA obtained in Example 1 was used as a template using clonetech's diversify PCR random mutagenesis kit (catalog #: 630703), and the mutagenesis reactions 4 (mutagenesis reactions 4) of Table III described in the instructions.
  • PCR was performed under the conditions. Specifically, PCR was performed using the primers of SEQ ID NOs: 2 and 4, and the cycle consisting of 30 seconds of denaturation at 94 ° C. and 30 seconds of elongation at 68 ° C. was repeated 25 times.
  • a randomized rpoD gene pool (mutated art rpoD DNA pool) into which the base substitution was randomly obtained was obtained as a PCR result, and the PCR result was digested with BamHI and EcoRI , and a 0.5Kb DNA fragment (hereinafter, "art rpoD m fragments ”) were obtained by electrophoresis on 0.8% agarose gel and then eluting.
  • Example 4 Construction of a recombinant vector pCC1BAC-rpoD mutant library comprising a mutated rpoD
  • the pCC1BAC-patial rpoD vector prepared in Example 2 was treated with restriction enzymes BamHI and EcoRI , followed by alkaline phosphatase (NEB).
  • Example 3 Thereafter, the art rpoD m fragments obtained in Example 3 were treated with restriction enzymes BamHI and EcoRI , respectively, and ligated with the restriction enzyme-treated pCC1BAC-patial rpoD to transform into TransforMax EPI300 Electrocompetent E.coli (EPICENTRE). Colonies were selected by incubating in LB medium (LB plate) containing 15 ⁇ g / ml chloramphenicol. The selected colonies were collected and plasmid prep was performed to prepare pCC1BAC-rpoD mutant library.
  • LB medium LB plate
  • the pCC1BAC-rpoD mutant library obtained in Example 4 was introduced by transformation into electro-competent cells of a threonine producing strain KCCM10541.
  • E. coli KCCM10541 (Korean Patent No. 10-0576342) used in this example is E. coli in which the galR gene is inactivated from E. coli KFCC10718 (Korean Patent No. 10-0058286), which is a strain for producing L-threonine.
  • the recombinant microbial library prepared in Example 5 was cultured in the threonine titer medium of Table 4 to confirm the improvement of L-threonine productivity.
  • E. coli KCCM10541 / pCC1BAC-rpoD and E. coli KCCM10541 / pCC1BAC-rpoD mutant library incubated overnight in LB solid medium in a 33 °C incubator, each one platinum in 25 mL titer medium of Table 4 , It was incubated for 48 hours in an incubator at 33 °C, 200 rpm. This process was repeated to evaluate the rpoD mutant library, and among them, clones given the improved yield were selected.
  • the parent strain KCCM 10541 and the control (control) strain KCCM 10541 / pCC1BAC-rpoD strain produced about 30.4 g / L of L-threonine when incubated for 48 hours. .
  • KCCM10541 / pCC1BAC-rpoD m19 with the most improved L-threonine production capacity among the transformed Escherichia coli was deposited as KCCM11560P on August 6, 2014 at the Korea Microorganism Conservation Center.
  • Example 6 several of the rpoD mutations in which the effect of improving the threonine production was confirmed, an experiment was conducted to reconfirm the effect thereof based on the wild type strain.
  • the rpoD mutation identified in Example 6 was transformed into wild type strain W3110 in the same manner as in Example 5, which was named W3110 / pCC1BAC-rpoD m .
  • the pACYC184-thrABC vector was introduced into the strain into which the rpoD mutation was introduced to confer threonine production capacity.
  • pACYC184-thrABC was produced in the following manner.
  • the genomic DNA of Escherichia coli KCCM 10541 (Korean Patent No. 10-0576342, Chinese Patent No. 100379851C), which is a strain for producing L-threonine derived from E. coli KFCC 10718 (Korean Patent No. 10-0058286), was used as a template. PCR reaction was carried out using the primers of 6, and the obtained DNA fragments were separated and purified, followed by purification by HindIII enzyme treatment to prepare thrABC DNA fragments (Table 6). The pACYC184 vector was purified and prepared by HindIII enzyme treatment, followed by ligation to prepare pACYC184-thrABC vector. In this way the produced vector introduced into the W3110 / pCC1BAC rpoD-m strain was produced W3110 / pCC1BAC-rpoD m, pACYC184 -thrABC strain.
  • Example 7 The recombinant microorganism prepared in Example 7 was cultured in a Erlenmeyer flask using the threonine titer medium of Table 7 to confirm the improvement of L-threonine productivity.
  • the wild type strains W3110 / pCC1BAC and W3110 / pCC1BAC-rpoD, W3110 / pCC1BAC-rpoD m2 and W3110 / pCC1BAC-rpoD m19 strains did not produce any L-threonine at 48 hours of incubation. However, it was confirmed that the consumption sugar was reduced in the case of variant introduction strain.
  • the W3110 / pACYC184-thrABC and pCC1BAC strains recombined to produce threonine on a wild type basis produced 1.42 g / L of L-threonine, and the W3110 / pACYC184-thrABC and pCC1BAC-rpoD strains were 1.43 g / L. Produced 2.8% yield.
  • a variation combination vector was prepared for several of the most selected variants.
  • rpoD m24 mutations have been introduced all mutations the amino acid sequence of 446th, 448th, 466th, 527th, 567th variation and mutation of 579th, 612th variation of rpoD m3 of rpoD m16.
  • rpoD m25 (SEQ ID NO: 33) was prepared by combining the 496th amino acid sequence of rpoD m15 with the 579th and 612 mutations of rpoD m1 .
  • rpoD m26 (SEQ ID NO: 34), which combines the 440th , 579, and 612th amino acid mutations with the most selected 440th, 579, and 612th amino acid mutations, and rpoD m27 (SEQ ID NO: 34).
  • rpoD m27 (SEQ ID NO: 34).
  • a combination of the low frequency of the selected mutations were also produced to confirm the effect.
  • 484th variation, 509th variation of rpoD m20 of the 477th variation and rpoD m19 of rpoD m17 rpoD m28 (SEQ ID NO: 36) production and, 459th mutation of 599th variation and rpoD m20 of rpoD m18, rpoD m29 (SEQ ID NO: 37) was prepared by combining the 576th mutation of rpoD m21 .

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Abstract

La présente invention concerne : un nouveau polypeptide à facteur sigma d'ARN polymérase variant 70 (σ70); un polynucléotide codant pour celui-ci; un microorganisme contenant le polypeptide; et un procédé de production de L-thréonine au moyen dudit microorganisme.
PCT/KR2015/009381 2014-09-05 2015-09-04 Microorganisme ayant une productivité améliorée de l-thréonine, et procédé de production de l-thréonine au moyen de celui-ci WO2016036209A1 (fr)

Priority Applications (8)

Application Number Priority Date Filing Date Title
CN201580006567.2A CN106029879B (zh) 2014-09-05 2015-09-04 具有提高的l-苏氨酸生产能力的微生物以及使用其生产l-苏氨酸的方法
BR112016015218-2A BR112016015218B1 (pt) 2014-09-05 2015-09-04 Microrganismo com capacidade melhorada de produção de l-treonina e método para produzir l-treonina com o uso desse
PL15837846T PL3144385T3 (pl) 2014-09-05 2015-09-04 Mikroorganizm o ulepszonej produktywności l-treoniny i sposób produkcji l-treoniny z jego wykorzystaniem
ES15837846T ES2820583T3 (es) 2014-09-05 2015-09-04 Microorganismo con productividad mejorada de l-treonina, y método de producción de L-treonina mediante el uso del mismo
US15/117,437 US10760108B2 (en) 2014-09-05 2015-09-04 Modified RNA polymerase sigma factor 70 polypeptide
EP15837846.3A EP3144385B1 (fr) 2014-09-05 2015-09-04 Microorganisme ayant une productivité améliorée de l-thréonine, et procédé de production de l-thréonine au moyen de celui-ci
US16/447,814 US10968467B2 (en) 2014-09-05 2019-06-20 Modified RNA polymerase sigma factor 70 polypeptide
US16/930,082 US11312982B2 (en) 2014-09-05 2020-07-15 Microorganism with improved L-threonine producing capability, and method for producing L-threonine by using the same

Applications Claiming Priority (4)

Application Number Priority Date Filing Date Title
KR20140119138 2014-09-05
KR10-2014-0119138 2014-09-05
KR10-2015-0125440 2015-09-04
KR1020150125440A KR101865998B1 (ko) 2014-09-05 2015-09-04 L-쓰레오닌 생산능이 향상된 미생물 및 이를 이용한 l-쓰레오닌 생산방법

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US15/117,437 A-371-Of-International US10760108B2 (en) 2014-09-05 2015-09-04 Modified RNA polymerase sigma factor 70 polypeptide
US16/447,814 Continuation US10968467B2 (en) 2014-09-05 2019-06-20 Modified RNA polymerase sigma factor 70 polypeptide
US16/930,082 Division US11312982B2 (en) 2014-09-05 2020-07-15 Microorganism with improved L-threonine producing capability, and method for producing L-threonine by using the same

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WO2019182413A1 (fr) 2018-03-23 2019-09-26 씨제이제일제당 (주) Granules contenant de l'acide l-aminé et leur procédé de préparation
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